The development of hybrid crop varieties has enabled an increase in crop productivity, mainly due to hybrid vigor and increased uniformity. Most crops show hybrid vigor, but commercial production of hybrids is only feasible if a reliable and cost-effective pollination control system is available; hybrid seed production requires a system that prevents unwanted self-pollination.
Methods that can be used to prevent self-pollination include mechanical removal of anthers or male flowers, application of male-specific gametocides, and use of genetic cytoplasmic or nuclear-encoded male sterility. Mechanical removal is an expensive practice, which also undesirably reduces crop yield due to plant damage. With respect to the use of cytoplasmic male-sterile (CMS) lines, these lines have a mutation in their mitochondrial genome, and thus the male sterility is inherited as a dominant, maternally-transmitted trait. Cytoplasmic male sterility requires CMS mutants and nuclear restorer available in a given crop. Perez-Prat and van Lookeren Campagne (2002) Trends Plant Sci. 7:199-203.
In view of the foregoing considerations, the production of commercial hybrid corn seed typically utilizes the planting of male and female inbred lines in separate rows or blocks in an isolated field to reduce the possibility of contamination. The female inbred is then detasseled before pollen shed, which ensures cross-pollination by the male inbred. Hybrid seed is harvested and processed from the ears of the cross-pollinated female inbred. Manual or mechanical detasseling contributes to the high cost of hybrid corn seed. Furthermore, hybrid seeds generally have lower yields, which further results in lower revenues and profitability.
RNA interference (RNAi) is a process utilizing endogenous cellular pathways, whereby an interfering RNA (iRNA) molecule (e.g., a dsRNA molecule) that is specific for a target gene sequence results in the degradation of the mRNA encoded thereby. In recent years, RNAi has been used to perform gene “knockdown” in a number of species and experimental systems; for example, C. elegans, plants, insect embryos, and cells in tissue culture. See, e.g., Fire et al. (1998) Nature 391:806-11; Martinez et al. (2002) Cell 110:563-74; McManus and Sharp (2002) Nature Rev. Genetics 3:737-47.
RNAi accomplishes degradation of mRNA through an endogenous pathway including the DICER protein complex. DICER cleaves long dsRNA molecules into short double-stranded fragments of approximately 20 nucleotides. The inhibitory double-stranded RNA is unwound into two single-stranded RNAs: the passenger strand and the guide strand. The passenger strand is degraded, and the guide strand is incorporated into the RNA-induced silencing complex (RISC). Post-transcriptional gene silencing (translational repression) occurs when the guide strand binds specifically to a complementary sequence of an mRNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex.
Plant micro-RNAs (miRNAs) are typically produced from fold-back structures having a partial double-stranded structure (e.g., “hairpins”), and usually are nearly perfectly complementarity with target sites, which are found most commonly in protein-coding regions of the genome. As a result, plant miRNAs function generally to guide mRNA cleavage. Watson et al. (2005) FEBS Lett. 579:5982-7. In contrast, animal miRNAs contain relatively low levels of complementarity to their target sites, and thus generally do not guide cleavage, but rather function to repress expression at the translational or co-translational level. Watson et al. (2005), supra; Tomari and Zamore (2005) Genes Dev. 19(5):517-29. Although miRNA sequences are not conserved between plants and animals, the RNAi pathways that utilize these genes are highly similar. Millar and Waterhouse (2005) Funct. Integr. Genomics 5:129-35. For example, while the biogenesis of miRNAs in plants is accomplished by a different set of related enzymes than accomplish the biogenesis of animal miRNAs, the miRNA molecules themselves have a characteristic structure that is capable of effecting mRNA cleavage or translational repression, depending on their degree of sequence complementarity to the target gene. Id.
In addition to miRNAs, plants also produce endogenous 21-25 nucleotide small inhibitory-RNAs (siRNAs). Most of these differ from miRNAs, in that they arise from double-stranded RNA (rather than imperfect fold-back structures), which in some cases are generated by the activity of RNA-Dependent RNA Polymerases (RDRs).
Most plants contain four DICER-LIKE (DCL) proteins, one of which (DCL1) is necessary for maturation of most miRNA precursors. Kurihara and Watanabe (2004) Proc. Natl. Acad. Sci. USA 101:12753-8. Animal miRNA precursor processing requires the sequential nucleolytic activity of DROSHA and DICER. Lee et al. (2003) Nature 425:415-9. In animals, Exportin-5 (ExpS) regulates the transport of pre-miRNAs from the nucleus to the cytoplasm. Bohnsack et al. (2004) RNA 10:185-91.
Only RNA transcripts complementary to the siRNA and/or miRNA are cleaved and degraded by RNAi, and thus the knock-down of mRNA expression is sequence-specific. The gene silencing effect of RNAi persists for days and, under experimental conditions, can lead to a decline in abundance of the targeted transcript of 90% or more, with consequent reduction in levels of the corresponding protein.